While the traditional focus of basic and clinical cancer immunology research has been on MHC class I-restricted cytolytic CD8+ T lymphocytes, our laboratory has innovated research into the role of MHC class II-restricted CD4+ T cells in human antitumor immunity. CD4+ cells are critically important for initiating, orchestrating and maintaining antitumor immune responses in animal models. This laboratory has been on the forefront of developing new technologies for the biochemical and molecular identification of tumor-associated proteins recognized by CD4+ T cells, including protein purification and cDNA library screening approaches targeting the MHC class II Ag processing/presentation pathway. Two recently completed clinical trials investigated tyrosinase, a melanoma-associated antigen recognized by both CD4+ and CD8+ T cells, as a therapeutic target in melanoma patients with stage IV disease. Despite evidence for effective immunization with recombinant poxvirus/tyrosinase vaccines plus systemically administered IL-2, objective clinical responses were few. Current projects, aiming to identify more optimal target molecules for immunotherapy and to modulate immune responses to therapeutic advantage, include:1) Evaluation of novel tumor antigens for clinical application. Following the recent finding that a single activating mutation in the signaling molecule BRAF is associated with >60% of melanomas, our laboratory has investigated whether mutant BRAF can be recognized by CD4+ T cells from melanoma patients. T cells specific for a mutant 29-mer peptide, restricted by diverse MHC class II alleles, have been generated and can recognize tumor cells harboring the mutation. Because its expression is tumor-specific and apparently required to maintain the malignant cell phenotype, mutant BRAF exemplifies targets that may be ideal for cancer immunotherapy.2) Characterizing mechanisms of endogenous MHC class II antigen processing. Some of the melanoma-associated antigens identified in our laboratory as being recognized by CD4+ T cells are processed through poorly understood endogenous intracellular pathways, rather than through the classical exogenous pathway. Studies are underway to define mechanisms by which these antigens (mutant versions of triosephosphate isomerase, BRAF, CDC27, and neo-poly[A] polymerse) are processed and presented for immune recognition. Such studies may lead to the design of more efficient vaccines through intracellular targeting. 3) Modulating the B7H1/PD-1 axis to enhance antitumor immunity. PD-1 is an inhibitory receptor on activated T cells which induces negative signaling upon ligation of B7-H1, a B7 family member expressed on melanomas and other human tumors. In murine models, interrupting the interaction of PD-1 and B7-H1 enhances tumor-specific immunity. Investigations aimed at modulating PD-1 expression and function on human T cells are currently in progress, and have direct relevance to ongoing clinical trials of vaccination and adoptive T cell transfer.